Low phosphate growth of fungal mycelia

ABSTRACT

Fungal mycelia are grown in a medium wherein the phosphate level is only slightly above the level which will limit growth, which results in a slightly lower content of ribonucleic acid in the mycelia but which results in the cells being much more amenable to removal of ribonucleic acid by simple means such as thermal shock.

BACKGROUND OF THE INVENTION

Recent concern for the welfare of the world population has includedconsideration of additional means for feeding the rapidly increasingnumber of people involved. The problem embraces providing both adequateper capita caloric intake and a balanced diet, with particular referenceto the lack of protein affording foods in many parts of the world. Onemeans for providing necessary protein supplies is through the growth offungal mycelia for use either as food or food supplements.

Production of edible fungal mycelia in large quantities may beaccomplished by fermentation processes employing a carbohydratesubstrate. The principal requirements are that the substrate material berelatively inexpensive and readily consumed by the selectedmicroorganism.

The human metabolic system produces uric acid in the metabolism ofribonucleic acid. Since man does not have a uricase enzyme system, uricacid is not further broken down and is excreted in the urine. Becauseuric acid salts have a very low solubility in biological fluids, theywill accumulate in the body in crystalline form if produced in largerquantities than the body can excrete. This may lead to the conditionknown as gout. For this reason nutritionists recommend that theribonucleic acid intake in diet be kept at a low level.

Fungal mycelia generally contain from 7 to 9% nucleic acids. If thefungal mycelia are to be used as a protein source in human feeding,nutritionists generally recommend that the amount of nucleic acidscontributed by single cell protein or fungal mycelia should not exceed 2grams per day.

A preferrred way of utilizing edible fungal mycelia is in the form ofwhole cells. Accordingly, there is a need for a means for removingnucleic acids from within the fungal mycelia. This desirably isaccomplished with a minimum loss of protein material from within thecells in order to maintain the nutritional attractiveness of such fungalmycelia. One method found to work well with some fungi in reducingnucleic acid content is the isothermal shock method disclosed in U.S.Pat. No. 4,041,189 Any procedure permitting increased effectiveness ofthis procedure should therefore decrease the possibility of gout whenprocessed fungal material is used as human food.

SUMMARY OF THE INVENTION

The present invention involves growing fungal mycelia in a mediumwherein the phosphate level is only slightly above the level which willlimit growth of the mycelia. The thus grown fungal mycelia have beenmore amenable to loss of ribonucleic acid upon processing.

DESCRIPTION OF THE INVENTION

According to the present invention there is provided a process for theproduction of edible protein containing fungal mycelia which comprisesincubating and proliferating, under aerobic conditions, non-toxicstrains of fungi in a culture medium containing essentialgrowth-promoting nutrient substances and separating the proliferatedmycelia.

The separated proliferated fungal mycelia after reduction of theribonucleic content thereof are suitable for use as a human foodstuff.The preferred fungi for use in the present invention are FungiImperfecti of the genus Fusarium with Fusarium graminearum Schwabe beingespecially preferred. The most preferred microfungus is Fusariumgraminearum Schwabe deposited with the Commonwealth MycologicalInstitute (I.M.I.) and the American Type Culture Collection and assignedthe numbers I.M.I. 145,425 and A.T.C.C. 20,334. Suitable reisolates ofthis microfungus also deposted with the Commonwealth MycologicalInstitute and the American Type Culture Collection include I.M.I.154,209, A.T.C.C. 20,329; I.M.I. 154,210, A.T.C.C. 20,333; I.M.I.154,211, A.T.C.C. 20,330; I.M.I. 154,212, A.T.C.C. 20,331; I.M.I.154,213, A.T.C.C. 20,332. Other suitable nontoxic microfungi include butare not limited to Fusarium Oxysporum (I.M.I. 154,214, A.T.C.C. 20,328),and Fusarium Solani (I.M.I. 154,217, A.T.C.C. 20,327), with the numbersof strains thereof which have been deposited with the CommonwealthMycological Institute and the American Type Culture Collection given inparenthesis.

The present invention is based on the discovery that when fungal myceliaare grown under aerobic conditions in a culture medium containingessentially growth-promoting nutrient substances wherein the myceliacells are partly starved of phosphate, the cells are more readilyamenable to the reduction of their ribonucleic acid (etc) content. Thenet effect is an enhanced reduction in residual nucleic acid in theproduct, thus a product with more desirable properties as a human foodsource.

Generally, the amount of phosphate present in the medium should be fromthe level where phosphate is the growth limiting factor in the fermentercomposition up to 5 millimolar above the growth limiting level. ForFusarium graminearum Schwabe A.T.C.C. 20,334 grown at 20 g/l. thisgrowth limiting level of phosphate has been found to be about 6millimolar. For this particular Fusarium a phosphate concentration inthe initial medium of from 7 to 10 millimolar has been found to give thebest results.

The preferred ribonucleic acid reduction is by enzyme activation. Thepreferred enzyme activation is by thermal shock. Generally, this is doneby maintaining the fungal mycelial mass in an aqueous suspension at a pHof from 4.7 to 7.0 at a temperature of about 64° for a time of about 15minutes. The process of this isothermal shock has been reported in U.S.Ser. No. 507,123 and is not claimed in this invention. The grown fungalmycelia are harvested, and filtered to remove growth medium, if desired.The fungal mycelia are then brought rapidly to the desired temperaturewith an aqueous buffer solution in the range of pH 4.7 to 7.0. Analternate approach would be to heat the whole mycelia culture slurryrapidly. The resulting treated fungal mycelia mass may then be harvestedagain, for example, by filtration and washing with water and thereafterformulated into foods. In the case of the preferred Fusarium graminearumSchwabe A.T.C.C. 20,334 ribonucleic acid content can readily be reducedto below 0.5 weight percent (5 milligrams per gram of mycelia dry basis)using this very easily carried out thermal enzyme activation.

EXAMPLES

A continuous 8-liter fermenter is sterilized and continuously chargedwith a sterile medium consisting of

    ______________________________________                                                          g per 100 liters                                            ______________________________________                                        MgSO.sub.4          50                                                        ZnSO.sub.4 . 7H.sub.2 O                                                                           0.83                                                      CuSO.sub.4 . 5H.sub.2 O                                                                           0.167                                                     MnSO.sub.4 . 1H.sub.2 O                                                                           0.63                                                      FeSO.sub.4 . 7H.sub.2 O                                                                           0.62                                                      K.sub.2 SO.sub.4    10.0                                                      (NH.sub.4).sub.2 SO.sub.4                                                                         144.0                                                     NaMoO.sub.4 . 2H.sub.2 O                                                                          0.083                                                     CoCl.sub.2 . 6H.sub.2 O                                                                           0.17                                                      NaCl                1.0                                                       CaCl.sub.2          8.0                                                       Biotin              0.0006                                                    Dextrose . H.sub.2 O (carbohydrate)                                                               5,000.0                                                   Ammonium citrate    4.0                                                       Boric acid          0.05                                                      KH.sub.2 PO.sub.4   as reported in Table I                                    Water               to 100 liters                                             ______________________________________                                    

The rate of charging the sterile medium is 1.3 liters per hour. Themedium is inoculated with a spore suspension of Fusarium graminearumSchwabe I.M.I. 145,425, A.T.C.C. 20,334. The fermenter is stirred with a6-bladed disc turbine operated at 750 rpm. Air is flowed through thefermenter at a rate of 3.6 liters per minute. Additional oxygen flow is2.0 liters per minute. The fermenter temperature is 29.2° C., and the pHis 4.8. Ammonia is added to control the pH and provide additionalmycelial nutrient. The fermenter productivity is about 3 gl⁻¹ hr⁻¹. Theproduct fungal mycelium is continuously drawn off the fermenter, andcollected in a product receiver which is held at 8° C. After steadyoperation is achieved the pooled product for about 10 hours operation isthen harvested. The resultant filter cake is then suspended in 8.0liters of filtrate which has been preheated to 72° C. and adjusted to pH6.0 with NaOH. Addition of the filter cake to the heated filtratedecreases the slurry temperature to 64° C. which is then maintained for20 minutes. This treatment serves to reduce the ribonucleic acid contentof the filter cake. The slurry is then filtered and washed on the filterbed with one half the bed volume of distilled water. The filter cake isthen dewatered with a vacuum to a solids content of 27.6 weight percent.At this point the wet filter cake comprises an entangled mass of limpflexible mycelial filaments. Samples of the fungal mycelia are analyzedfor α-aminonitrogen and ribonucleic acid before and after reduction ofribonucleic acid (RNA). The "yield" reported in Table I is determined bydividing the fermenter cell mass by the glucose used. Typical controlrun yields are about 0.5.

                                      TABLE I                                     __________________________________________________________________________                                    Fresh A3/5    RNA reduced                     Initial Concen.   Approx. Residual                                                                       Typical                                                                            Exfermenter                                                                           Cell Mass                                                                           product Mg/g                    Code Phosphate, mM                                                                              Phosphate                                                                              Run hour                                                                           αAN                                                                         RNA g/l   αAN                                                                         RNA     Yield               __________________________________________________________________________    Control                                                                            28mM         (21)       85 69.5                                                                              90.0                                                                              29    72  16.                         Run 1                                                                              7.0          2          62 64.3                                                                              88.4                                                                              20.1                                       4.0          <1         85 59.6                                                                              63.2                                                                              15.9                                       6.0          1         103 64.9                                                                              77.1                                                                              19.4                                       6.0          <1        111 50.9                                                                              52.1                                                                              27.0  67.9                                                                              3.5     0.49                     8.0          2         158 62.0                                                                              75.3                                                                              24.7              0.52                     7.0          1         203 61.2                                                                              70.0                                                                              23.5  70.7                                                                              2.8     0.50                     7.5          2         213 62.8                                                                              79.4                                                                              22.3  69.7                                                                              2.2     0.52                     9.0          3         230 62.8                                                                              80.0                                                                              24.4  70.5        0.53                     6.0          1         248 61.7                                                                              74.6                                                                              20.1  66.6                                                                              3.4     0.50                     28.0                                                                              (starting at                                                                           23        278 64.4                                                                              85.6                                                                              20.8  68.2                                                                              6.7 (rising)                         hour about 265)                                                      Run 2                                                                              7.0          2.5        81 61.7                                                                              80.1                                                                              17.6  68.6                                                                              3.7                              7.0          2.5       155 63.5                                                                              82.1                                                                              17.0  69.0                                                                              4.9                         __________________________________________________________________________

As can be seen the use of low phosphate in the fermenter in accordancewith the present invention results in a greatly lowered amount ofribonucleic acid in the treated product without a significant loss ofprotein as evidenced by the α-aminoacid analysis (αAN).

I claim:
 1. A process comprising growing an edible strain of Fusarium ona carbohydrate substrate under aerobic conditions in a culture mediumcontaining essential growth-promoting nutrient substances to produce amycelial mass, wherein the phosphate present in the medium is maintainedat from the amount which is growth limiting up to 5 millimolar above theamount which is growth limiting, harvesting at least a portion of thegrown fungal mycelial mass and reducing the ribonucleic acid content ofthe harvested fungal mycelial mass by means of enzyme activation.
 2. Theprocess of claim 1 wherein the reduction of ribonucleic acid is effectedby maintaining the fungal mass in an aqueous suspension at a pH between4.7 and 7.0 at a temperature between 55° and 72° C. for a time of atleast 60 seconds.
 3. The process of claim 2 wherein the strain ofFusarium is a strain of Fusarium graminearum Schwabe, Fusariumoxysporum, or Fusarium solani.
 4. The process of claim 3 wherein thestrain of Fusarium is a strain of Fusarium graminearum Schwabe.
 5. Theprocess of claim 4 wherein the concentration of the strain of Fusariumis maintained at from 15 to 25 grams per liter and the phosphate levelin the fermenter is maintained at from 7 millimolar to 10 millimolar. 6.The process of claim 5 wherein the ribonucleic acid reduction step isperformed by rapidly raising the temperature of the fungal mycelial massat least 25° C. to from 55° to 72° C. and a product containing less than0.5 weight percent ribonucleic acid is obtained.
 7. The process of claim6 wherein the Fusarium graminearium Schwabe is derived from strainA.T.C.C. 20,344.